Hydroxychloroquine and a low antiresorptive activity bisphosphonate conjugate prevent and reverse ovariectomy-induced bone loss in mice through dual antiresorptive and anabolic effects

Osteoporosis remains incurable. The most widely used antiresorptive agents, bisphosphonates (BPs), also inhibit bone formation, while the anabolic agent, teriparatide, does not inhibit bone resorption, and thus they have limited efficacy in preventing osteoporotic fractures and cause some side effects. Thus, there is an unmet need to develop dual antiresorptive and anabolic agents to prevent and treat osteoporosis. Hydroxychloroquine (HCQ), which is used to treat rheumatoid arthritis, prevents the lysosomal degradation of TNF receptor-associated factor 3 (TRAF3), an NF-κB adaptor protein that limits bone resorption and maintains bone formation. We attempted to covalently link HCQ to a hydroxyalklyl BP (HABP) with anticipated low antiresorptive activity, to target delivery of HCQ to bone to test if this targeting increases its efficacy to prevent TRAF3 degradation in the bone microenvironment and thus reduce bone resorption and increase bone formation, while reducing its systemic side effects. Unexpectedly, HABP-HCQ was found to exist as a salt in aqueous solution, composed of a protonated HCQ cation and a deprotonated HABP anion. Nevertheless, it inhibited osteoclastogenesis, stimulated osteoblast differentiation, and increased TRAF3 protein levels in vitro. HABP-HCQ significantly inhibited both osteoclast formation and bone marrow fibrosis in mice given multiple daily PTH injections. In contrast, HCQ inhibited marrow fibrosis, but not osteoclast formation, while the HABP alone inhibited osteoclast formation, but not fibrosis, in the mice. HABP-HCQ, but not HCQ, prevented trabecular bone loss following ovariectomy in mice and, importantly, increased bone volume in ovariectomized mice with established bone loss because HABP-HCQ increased bone formation and decreased bone resorption parameters simultaneously. In contrast, HCQ increased bone formation, but did not decrease bone resorption parameters, while HABP also restored the bone lost in ovariectomized mice, but it inhibited parameters of both bone resorption and formation. Our findings suggest that the combination of HABP and HCQ could have dual antiresorptive and anabolic effects to prevent and treat osteoporosis.


INTRODUCTION
Osteoporosis is a major disease of aging characterized by decreased bone mass and strength, resulting in increased fracture risk.About 44 million people in the U.S. have low bone mineral density (BMD), and 10 million of them are osteoporotic. 1Elderly patients with femoral neck fractures develop serious complications, including pneumonia and deep vein thrombosis due to prolonged bed rest, and up to 20% of them die within the 12 months after the fracture. 2steoporosis is caused by imbalanced bone remodeling, due to enhanced bone resorption and relatively reduced bone formation, mediated by osteoclasts (OCs) and osteoblasts (OBs) respectively. 1oth antiresorptive and anabolic agents are available, but they do not satisfactorily treat osteoporosis.For example, the antiresorptive agents, bisphosphonates (BPs) and the RANKL monoclonal antibody, denosumab, also inhibit bone formation, 3,4 but they reduce the rate of osteoporotic fracture by only ~50% 5 and a small percentage of patients also develop osteonecrosis of the jaw or atypical femoral shaft fractures. 6In addition, following discontinuation of denosumab some patients have vertebral fractures due to rebound increased bone resorption. 7,8Furthermore, treatment with the anabolic agent, teriparatide, is limited to two years due to safety concerns, [9][10][11][12] and is typically followed by treatment with antiresorptive agents, 13 because their anabolic effects are transient, and discontinuation results in increased bone resorption. 14In addition, combination therapy with teriparatide and a BP does not appear to offer advantages over the use of either agent alone. 15,16Similarly, switching from alendronate to teriparatide does not improve hip BMD, although addition of an anabolic agent to ongoing alendronate treatment does. 17The recently approved sclerostin monoclonal antibody, romosozumab, transiently increases bone formation and inhibits bone resorption in osteoporotic patients. 18However, treatment is associated with severe side effects, including myocardial infarction, stroke and cardiovascular death, 11 and is recommended for up to only 1 year in patients with severe osteoporosis. 19Therefore, there is an unmet need to develop a new strategy to prevent and treat osteoporosis.
BPs are characterized by two phosphate groups joined to a central carbon atom forming a stable, degradation-resistant phosphomethylene moiety with two potential side chains (R1 and R2).For most of the clinically used BPs, including zoledronate, alendronate, ibandronate, risedronate, and pamidronate, one side chain is -OH, which adds binding affinity to bone mineral, and another side chain introduces a basic nitrogen moiety via an alkyl chain or a heterocyclic group to enhance their antiresorptive activity. 20Consequently, these are called the nitrogen-containing BP (N-BP) class of drugs.It is now clear that the N-BPs inhibit farnesyl pyrophosphate synthase (FPPS), 21,22 in particular in osteoclasts (OCs), resulting in disruption of cytoskeletal organization, loss of OC ruffled border formation, altered vesicular trafficking, and apoptosis. 23The distance and orientation of the nitrogen moiety relative to the phosphonate groups (for example, the length of the aminoalkyl chain or the position of the nitrogen atom within a heterocyclic ring) markedly influence the antiresorptive potency of N-BPs. 24By virtue of calcium-chelating properties, the phosphonate groups of BPs can deposit tightly in bone, and thus a druggable agent that is linked to a BP can be targeted for delivery to bone specifically to treat bone diseases more efficiently, while reducing systemic side effects of the drugs. 25,26A group of BP analogs, for example, hydroxyalklyl BP (HABP), have been developed for the specific use for targeting druggable agents to bone. 25,26Our aim was to develop a BP with low antiresorptive activity conjugated to therapeutic agents with potential dual antiresorptive and anabolic effects, such as hydroxychloroquine (HCQ), and target them to bone to treat osteoporosis.
HCQ, an anti-inflammatory drug that is used to treat malaria and rheumatoid arthritis, could potentially be a dual antiresorptive and anabolic agent by preventing the lysosomal degradation of TNF receptor-associated factor 3 (TRAF3), an adaptor protein that transduces intracellular signaling of cytokines, including RANKL, 27,28 TNFα, 27,28 CD40L 29 and TGFβ30.TRAF3 negatively regulates OC formation by limiting non-canonical processing of NF-κB p100 to p52 in OC precursors, 27,28 and maintains OB differentiation by limiting TGFβ-induced GSK3β-mediated β-catenin degradation in OB precursors. 30Conditional knockout (cKO) of TRAF3 in either myeloid or OB lineage cells resulted in early onset osteoporosis in mice due to increased bone resorption and reduced bone formation, respectively. 30,31However, HCQ can have systemic side effects, such as cardiac toxicity and blindness, in up to 0.5% of patients. 32,33We therefore sought to link HCQ to a BP analog with low antiresorptive activity with the goal to administer an overall lower dose of HCQ as a long-term treatment of osteoporosis, while delivering a relatively high concentration to bone and thus reducing its systemic side effects because the BP would target HCQ to bone where it would be released in the acidic microenvironment of resorption lacunae at a locally higher concentration.HCQ was covalently linked to a BP analog, HABP, as in the synthesis of a bone-targeted chloroquine conjugate. 25nexpectedly, the compound was found to exist as a salt in water, composed of deprotonated HABP anions and protonated HCQ cations (Fig. S1 to S5).However, it is interesting that this combination of HCQ and HABP effectively prevented bone loss and restored the bone lost in ovariectomized mice due to antiresorptive and anabolic effects mediated by HABP and HCQ, respectively.HCQ alone, even at high dose, did not prevent or treat ovariectomy-induced bone loss, although it stimulated bone formation.The HABP alone restored the bone lost in ovariectomized mice by inhibiting bone resorption, but like other BPs, it also inhibited bone formation.Thus, a combination of low dose HCQ and HABP could be effective as a long-term treatment of osteoporosis by inhibiting bone resorption and stimulating bone formation simultaneously.

HABP-HCQ inhibits osteoclast formation and stimulates osteoblast differentiation in vitro
Bone marrow (BM) cells were cultured with M-CSF for 2 days to generate OC precursors (OCPs), which are macrophages that tightly attach to the culture dishes.10 ng/mL RANKL and different doses of HCQ, HABP obtained from the hydrolysis of the phosphonate ester tetraethyl HABP (Fig. 1a), or combinations of them were then added to the OCP cultures (after removing the suspended cells) in the presence of M-CSF for 3 days, following our published procedures. 34,35TRAP staining was performed to evaluate OC formation.Like HCQ, HABP-HCQ inhibited RANKLinduced OC formation from 1 μmol/L and completely suppressed OC formation at 10 μmol/L (Fig. 1b).When the cultures were stopped early (RANKL treatment for 2.5 d), we found that OC numbers and areas in the wells treated with 10 μmol/L HABP-HCQ were higher than those treated with the same dose of HCQ (Fig. 1b).HABP alone did not inhibit OC formation at 1 or 3 μmol/L, but completely inhibited OC formation at 10 μmol/L (Fig. 1b).
BM cells from C57BL/6 mice were cultured with stromal cell expanding medium for 5 days to expand stromal cells (SCs) attached to the culture dishes. 34,35These BM SCs were then induced for OB differentiation in the presence of HCQ or HABP-HCQ.Interestingly, the concentration of HCQ and HABP-HCQ that stimulated OB differentiation was lower than that required to inhibit OC formation, starting around 0.03 μmol/L (Fig. 1c).However, higher concentrations of HCQ (3 μmol/L) tended to inhibit OB differentiation (Fig. 1c), and this inhibition was attenuated by the combination of HCQ and HABP (Fig. 1c) because the HABP also slightly promoted OB differentiation from the BM stromal cells (Fig. 1d).

HABP-HCQ prevents PTH-induced osteoclast formation and BM fibrosis in vivo in mice
Intermittent single daily injections of PTH, like clinically used teriparatide, stimulate bone formation by downregulating SOST/ sclerostin expression in osteocytes, enabling the anabolic effect of Wnt signaling to proceed. 36In contrast, multiple daily injections of PTH that cause a sustained increase in PTH, like hyperparathyroidism, cause significant bone erosion due to enhanced RANKLinduced OC formation. 36We evaluated the dose of HABP-HCQ required to inhibit OC formation induced by multiple daily injections of PTH into the subcutaneous tissues overlying the calvarial bones of C57BL/6 mice 31 (Fig. 3a).As expected, multiple daily PTH injections significantly increased OC numbers in the calvarial BM and suture area (Fig. 3b upper panel).Neither 6 nor 30 μmol/L/kg HCQ inhibited PTH-induced OC formation in calvarial bones (Fig. 3b upper panel), suggesting that >30 μmol/L/kg HCQ is required to inhibit OC formation.In contrast, HABP-HCQ significantly inhibited PTH-induced OC formation in calvarial bones at 6 and 30 μmol/L/kg, but not at 1.25 μmol/L/kg (Fig. 3b upper panel), suggesting that ~6 μmol/L/kg HABP-HCQ is required to effectively inhibit PTH-induced OC formation in mice.
Multiple daily injections of PTH also cause BM fibrosis. 31HCQ (31.25 and 156, but not 6.25 μmol/L/kg) blocked PTH-induced BM fibrosis (Fig. 3b lower panel).In contrast, HABP-HCQ (from 6.25 μmol/L/kg) blocked PTH-induced BM fibrosis (Fig. 3B lower panel).Interestingly, the multilayered proliferation of fibroblastic/spindle cells on tibial trabecular surfaces in mice treated with multiple daily doses of PTH was prevented by 156 μmol/L/kg HCQ and 31.25 μmol/L/kg HABP-HCQ, and the mice had only a single layer of osteoblastic cells on trabecular surfaces (Fig. 3b lower panel).We also evaluated the effect of the HABP on PTH-induced OC formation and BM fibrosis and found that it inhibited PTH-induced OC formation in the calvarial bones, starting ~5 μmol/L/kg, a dose comparable to that of HABP-HCQ to inhibit OC formation (Fig. 3c upper panel).However, the HABP did not inhibit PTH-induced BM fibrosis (Fig. 3c lower panel).The dose of HCQ (≤5 mg/kg) prescribed for long-term treatment of rheumatoid arthritis and other autoimmune diseases in humans 37 corresponds to 26.4 mg (60 μmol/L)/kg in mice. 38We administrated 6.25 μmol/L/kg HABP-HCQ (containing 2.6 mg/kg HCQ) or 31.25 μmol/L/kg (containing 13.6 mg/kg) HCQ as starting doses to test if the lower dose of HABP-HCQ could prevent OVX-induced bone loss and if this depends on TRAF3 expression in WT (TRAF3 f/f ) and myeloid cell TRAF3 conditional knockout (TRAF3 f/f LyM cre , TRAF3 cKO) mice. 31As expected, OVX significantly decreased trabecular bone mass in the vertebrae of WT (TRAF3 f/f ) mice due to reduced Tb.N and Tb.Th compared to sham WT mice (Fig. 4a).
Consistent with the general concept that postmenopausal osteoporosis is characterized by high bone turnover, OVX resulted in increased OC numbers and surfaces as well as increased OB surfaces in vertebral bone sections from WT mice (Fig. 4b, c).Five doses/week of 6.25 μmol/L/kg HABP-HCQ significantly increased vertebral bone mass in ovariectomized WT mice to values that were similar to those in sham mice (Fig. 4a).Interestingly, HABP-HCQ not only significantly reduced OC numbers and surfaces (Fig. 4b), but also maintained OB surfaces at a high level in vertebral sections from ovariectomized mice (Fig. 4c).However, a 5-fold higher dose (31.25 μmol/L/kg) of HCQ did not increase vertebral BV/TV in the ovariectomized WT mice (Fig. 4a) probably because it did not reduce OC numbers or surfaces (Fig. 4b), although it maintained increased OB surfaces in the ovariectomized mice (Fig. 4c).
As a positive antiresorptive control, weekly injections of 150 μg (0.55 μmol/L)/kg zoledronic acid significantly increased vertebral BV/TV, Tb.N and Tb.Th (Fig. 4a) in ovariectomized WT mice.Of note, some large osteoclasts were present on the trabecular surfaces and in the BM space in zoledronic acid-treated mice (Fig. 4b), which have been reported in animals, 39 and are similar to the non-functional, "giant osteoclasts" described in bone samples from humans treated with bisphosphonates. 40We concluded that these giant osteoclasts in zoledronic acid-treated mice were nonfunctional because bone surfaces with active resorption lacunae were reduced (Fig. 4b).This is consistent with the report that bisphosphonates blocks prenylation of small GTPases, 41 causing disruption of cytoskeletal organization, vesicle transport, loss of the sealing zone, and ruffled border, 42,43 resulting in the detachment of osteoclasts from bone surfaces. 39Zoledronic acid also significantly reduced OB surfaces (Fig. 4c), as expected.As a positive anabolic control, intermittent PTH did not increase BV/TV in WT ovariectomized mice at this stage, probably because the OVX mice already had increased OB surfaces, and intermittent PTH did not further increase these (Fig. 4c) and did not change OC parameters either (Fig. 4b).
BV/TV values in the mice with TRAF3 cKO in myeloid cells were significantly lower than those in WT control mice (Fig. 4a), as expected.OVX further decreased BV/TV values (Fig. 4a), but it did not further increase OC numbers or eroded surfaces (Fig. 4b) in TRAF3 cKO mice likely because these resorptive parameters were already elevated.OVX did not change OB surfaces in TRAF3 cKO mice (Fig. 4c).As in the WT ovariectomized mice, HABP-HCQ, but not HCQ alone, significantly increased vertebral BV/TV values, associated with increased Tb.N and Tb.Th and reduced OC numbers and eroded surfaces in the ovariectomized TRAF3 cKO mice (Fig. 4b).However, vertebral BV/TV values in the ovariectomized TRAF3 cKO mice treated with HABP-HCQ were still markedly lower than those in WT mice.In addition, neither HCQ nor HABP-HCQ changed OB surfaces in the ovariectomized TRAF3 cKO mice (Fig. 4c).These findings suggest that HABP-HCQ-induced elevation of bone mass is partially dependent on the expression of TRAF3.This is consistent with the findings that HABP-HCQ prevents TRAF3 degradation induced by RANKL and TGFβ1 in myeloid cells and MPCs, respectively (Fig. 2a, b).Weekly doses of 150 μg (0.55 μmol/L) zoledronic acid did not reduce the number of giant OCs in the TRAF3 control floxed mice (Fig. 4b), but similar to its effect in the ovariectomized TRAF3 cKO mice, it reduced OB surfaces in the floxed control mice (Fig. 4c).Intermittent PTH significantly increased spinal BV/ TV values in the ovariectomized TRAF3 cKO mice (Fig. 4a), associated with increased OB surfaces (Fig. 4c), but it did not change OC parameters (Fig. 4b) because the OVX floxed mice already had increased OC surfaces and PTH did not further increase them (Fig. 4c).

HABP-HCQ reverses lost bone in ovariectomized mice by inhibiting bone resorption and increasing bone formation
We tested the effect of the HABP-HCQ on established bone loss.5 weeks post OVX, bone loss was established in mice, as confirmed by micro-CT analysis (Fig. 5a).We then treated the mice with HCQ, three doses of HABP-HCQ, HABP, or intermittent PTH for 5 weeks.HCQ, even given at high dose (20 μmol/L/kg), did not increase vertebral bone mass.HABP-HCQ dose-dependently restored the lost bone in ovariectomized mice to or above the level in sham mice (Fig. 5a).HABP (1.8 μmol/L/kg) and intermittent PTH also restored the lost bone in the OVX mice to the level in sham mice (Fig. 5a).
OVX increased OC numbers (Fig. 5b) and surfaces (Fig. S6), but HCQ did not change these parameters (Fig. 5b).In contrast, all doses of HABP-HCQ markedly reduced OC numbers (Fig. 5b) and surfaces (Fig. S6) in the ovariectomized mice, but their effects were lower than those of HABP alone (Figs. 5b and S6).As expected, intermittent PTH did not reduce OC numbers (Fig. 5b) or surfaces (Fig. S6).OB surfaces were increased 4 weeks after OVX and returned to the level of sham mice following 5 weeks of treatment (9 weeks after OVX) with vehicle (Fig. 5c).Interestingly, HCQ and the lowest dose of HABP-HCQ significantly increased OB surfaces in ovariectomized mice, while the highest dose of HABP-HCQ did not change OB surfaces (Fig. 5c).In contrast, HABP markedly reduced OB surfaces (Fig. 5c).As a positive control, intermittent PTH significantly increased OB surfaces in the ovariectomized mice (Fig. 5b).Like HCQ, HABP-HCQ also markedly increased dynamic bone formation parameters, including double-labeled surfaces, mineralizing surfaces, MARs and BFRs in the ovariectomized mice (Fig. 5d).HABP alone decreased, while intermittent PTH increased these dynamic parameters of bone formation (Fig. 5d), as expected.

DISCUSSION
Here, we report a dual anabolic and antiresorptive effect of a combination of HABP and hydroxychloroquine (HCQ) that effectively prevents ovariectomy-induced vertebral trabecular bone loss (Fig. 4) and restores the lost bone in ovariectomized mice to the level of sham mice (Fig. 5).We attribute the antiresorptive effects of HABP-HCQ primarily to HABP, rather than HCQ, because: (1) the lowest effective dose in vitro of HCQ (3 μmol/L) that inhibited OC formation is high and it would have been necessary to deliver very large doses of the drug to reach this threshold dose in vivo in mice; (2) HABP, but not HCQ, reduced bone resorption parameters in ovariectomized mice or in the multiple PTH injection model.We attribute the anabolic effects of HABP-HCQ to HCQ because: (1) the dose of HCQ to stimulate OB differentiation in vitro is as low as 0.03 μmol/L and the combination of HCQ and HABP is better than HCQ alone to stimulate OB differentiation in vitro (Fig. 1c, d); (2) the combination of HCQ and HABP, like HCQ alone, increased bone formation parameters in ovariectomized mice in vivo (Fig. 5); and (3) HABP alone strongly inhibited bone formation parameters in vivo (Fig. 5), although it slightly increased OB differentiation from BM stromal cells in vitro (Fig. 1d), like nitrogen-containing BPs. 44ur original purpose was to synthesize a single HABP-HCQ compound through a biodegradable linker to target delivery of HCQ to bone and release it slowly when the linker is degraded in the acid microenvironment generated by OCs.By this mechanism, the HCQ can be concentrated in bone specifically and thus the potential side effects of HCQ administered systemically could be avoided for long-term use to treat osteoporosis.Although HCQ dissociated from HABP when the conjugate was dissolved in water, HCQ associates with HABP as a salt (Fig. S3) and thus can circulate together to bone.Nevertheless, the combination of HABP and HCQ is more effective at preventing or restoring bone loss after ovariectomy than either alone.Further studies are required to determine if the HABP deposited in bone can remain associated with HCQ to prolong the half-life of HCQ in bone.
Our findings suggest that the administered dose of HABP, rather than having no or minimal antiresorptive effects, unexpectedly inhibited bone resorption, but the mechanism is different from that of conventional nitrogen-containing BPs (N-BPs), like zoledronate, which impair OC resorptive functions and accelerate OC apoptosis, also increase OC numbers 45 because they induce an acute phase reaction and inhibit FPPS, 21,22 resulting in disruption of cytoskeletal organization, loss of the ruffled border membrane, and altered vesicular trafficking. 23In contrast, the HABP inhibited OC formation, supported by our finding that it reduced OC numbers in vivo (Fig. 5b) without causing giant apoptotic OCs, as commonly occurs in animals (Fig. 4b) and humans treated with N-BPs. 45The effect of N-BPs to inhibit bone formation is attributed to their induction of farnesyl pyrophosphate accumulation in OBs due to the inhibition of FPPS activity 46 or is secondary to lower bone turnover.The HABP, in which one of the double side chains attached to the central carbon atom was replaced by one amido methylene chain, also inhibited bone formation, but we do not know if it inhibits FPPS activity.However, an amide function, rendering a nitrogen non-basic, further reduces any FPPS effect of a nitrogen in a bisphosphonate chain. 47,48The mild antiresorptive action of HABP appears to have enabled HCQ to better exert its anabolic function.This is different from the effects of N-BPs, such as alendronate and zoledronate, that strongly inhibit bone resorption and bone formation, thus disabling the effects of an anabolic agent. 15,17A high dose of HABP could potentially attenuate the anabolic effect of HCQ, as supported by our findings that the highest dose (20 μmol/L/kg) of HABP-HCQ reduced OB surfaces in ovariectomized mice to the levels in vehicle-treated controls, despite restoring the lost bone along with high bone formation parameters.Thus, further studies will be required to determine the optimal combined dose of HABP and HCQ to increase bone formation and inhibit resorption in ovariectomized mice.
One of the major mechanisms for the dual antiresorptive and anabolic effects of HABP-HCQ in preventing and treating osteoporosis (Figs. 4 and 5) is to stabilize TRAF3, which limits OC formation and maintains OB differentiation. 28,30,31HABP-HCQ works similarly to HCQ to stabilize TRAF3 in both myeloid cells and MPCs, and thus it inhibits OC formation and stimulates OB differentiation, respectively, in vitro (Fig. 2a, b).However, the lowest dose of HCQ that inhibits OC formation in vitro is higher than can be administered in vivo without serious adverse effects, and thus the dose of HCQ we administered did not inhibit bone resorption parameters in vivo.The myeloid cell-specific TRAF3 cKO mice we used in this study have enhanced parameters of bone resorption, with normal bone formation (Fig. 4), which is consistent with our previous reports. 27,31HABP-HCQ, like zoledronic acid, inhibited bone resorption in these TRAF3 cKO mice because the HABP component of HABP-HCQ exerts most of the antiresorptive action of the combination, partially dependent of TRAF3 in vivo.The HABP also prevented RANKL-induced TRAF3 degradation in OC precursors (Fig. 2a), and this mechanism may be related to its stimulation of OB differentiation in vitro (Fig. 2b).
Tissue fibrosis, characterized by the accumulation of extracellular matrix components, is a vital component of wound healing and tissue repair in response to injury, but it also occurs commonly in the elderly as a consequence of low-level of chronic inflammation of aging, particularly in fibrotic cardiac and respiratory diseases. 49BM fibrosis also occurs during aging, 50 associated with osteoporosis and defective WNT1 signaling, characterized by increased reticulin deposition and altered granulopoiesis. 51However, the fibrosis is not correlated with the severity of osteoporosis. 51Inhibition of BM fibrosis could be one of the mechanisms whereby the HABP-HCQ prevents and reverses ovariectomy-induced bone loss, although there are no published data showing a relationship between BM fibrosis and osteoporosis during aging.
In summary, a low dose of HCQ in combination with HABP has a dual anabolic and antiresorptive action, and thus prevents ovariectomy-induced bone loss in mice and restores the established bone loss caused by ovariectomy to normal levels.It is possible that this combination could be used long-term and safely for the prevention and treatment of osteoporosis by avoiding the systemic side effects of a high dose of including cardiac toxicity and blindness, or of a potent nitrogen-containing bisphosphonate, such as jawbone necrosis or atypical femoral shaft fractures.

Reagents
Recombinant murine M-CSF and RANKL were purchased from R&D Systems (Minneapolis, MN).Hydroxychloroquine sulfate and zoledronic acid were purchased from Sigma.Recombinant human PTH (1-34) was purchased from GenScript.Abs against TRAF3 and actin were purchased from Santa Cruz.Synthesis of HABP An oven-dried round bottom flask was charged with a solution of tetraethyl [(4-hydroxybutanamido)methylene)bis(phosphonic acid)] 3 in anhydrous dichloromethane (0.4 g, 1.028 mmol in 2 mL).After cooling to 0 °C, neat bromotrimethylsilane (0.8 mL) was added dropwise and the contents were carefully warmed to room temperature and stirred for 14 h.After concentrating under reduced pressure, the crude mixture was redissolved in methanol (2 mL) and concentrated.The procedure was repeated twice to yield 0.27 g (94.7%) of BP [(4-hydroxybutanamido)methylene) bis(phosphonic acid)] as a white waxy paste.A multiple PTH injection resorption-inducing model was used to determine the effective dose of HCQ and HABP-HCQ to inhibit OC formation in vivo.Briefly, 2-month-old female C57BL/6 mice were given vehicle, HCQ or HABP-HCQ daily for 6 days via intraperitoneal injection, and 80 μg (19.4 nmol/L) /kg of recombinant PTH (1-34), 4 times per day, during the last 3 days (day 4 to 6) via SC injection over calvariae.The mice were euthanized on day 7 (last PTH injection was given 2 h before death), and the calvarial and tibial bones were collected for processing and paraffin-embedded sectioning for TRAP and H&E staining to evaluate OC formation and BM fibrosis, respectively.The severity of fibrosis was evaluated by calculating the ratio of fibrosis tissue area to the trabecular area.
An ovariectomy (OVX) model was used to test if a low dose of HABP-HCQ is more effective than HCQ to prevent OVX-induced bone loss and if its effects depend on TRAF3 expression by OCs.3month-old female mice with TRAF3 conditional knockout (cKO) in myeloid cells (TRAF3 f/f LyM cre ; TRAF3 cKO) and their littermate control (TRAF3 f/f ) mice 31 were randomly divided into 6 groups, 8-9 mice/group.One group had sham surgery and 5 groups of mice for each phenotype had OVX surgery, and were randomly assigned to vehicle, HCQ (20 μmol/L/kg), HABP-HCQ (6 μmol/L/kg), zoledronic acid [Zol, 150 μg (0.55 μmol/L)] or intermittent PTH [80 μg (19.4 nmol/L)/kg daily injection] treatment, starting on the 2 nd day after surgery.Vehicle, HCQ, or HABP-HCQ in a volume of 0.1 mL per 10 g body weight were given via daily IP injection, 5 doses/week for 5 weeks.Zol was given as a single IP injection weekly, while PTH was given as daily supra-calvarial SC injections, 5 doses/week.To determine if a low dose of HABP-HCQ can treat established OVXinduced bone loss, 3-month-old female C57BL/6 mice were ovariectomized and subsequently given different doses of HABP-HCQ, HCQ, HABP or PTH for 4 weeks from the 5 th week postsurgery when bone loss in the vertebrae of OVX mice was confirmed by micro-CT.The mice were given injections of calcein (5 mg/kg) on the day 5 and 1 before sacrifice, following our standard protocol to assess bone formation parameters. 34,35After the mice were euthanized, the spinal bones were collected for micro-CT scanning, followed by histologic analysis of OBs and OCs in paraffin-embedded sections or dynamic bone formation parameters in plastic-embedded sections.

Micro-CT evaluation
Spines including the lower part of thoracic to upper coccygeal vertebrae were fixed in 10% neutral phosphate-buffered formalin for 48 h and were transferred to 70% ethanol at 4 °C for storage.The spines were scanned using a vivaCT 40 instrument (Scanco Medical) at a voxel size of 7 µm, 50 kVp, 144 µA and 800 ms integration time.The machine was set at a threshold of 220 to distinguish bone from soft tissues.Cancellous bone in L1 vertebrae was assessed in 300 transverse slices to determine bone volume [(BV/TV)/%], trabecular thickness (Tb.Th/μm), trabecular number (Tb.N/mm -1 ), trabecular separation (Tb.Sp/μm), according to standard guidelines. 52ne histomorphometric analysis After micro-CT scanning, L3-L5 vertebrae were decalcified for 3 weeks using 10% EDTA at 4 °C, processed, and embedded in paraffin.3-µm-thick sections at the center of the vertebrae with fewest trabeculae, selected from a series of continuously cut sections to ensure the measurement of all the sections was comparable, were blindly quantified for histomorphometric parameters, including the structural trabecular bone parameters, (BV/TV)/%, Tb.Th/μm, Tb.N/mm -1 , Tb.Sp/μm, and OB surfaces on H&E-stained sections, and OC parameters on TRAP-stained sections using an OsteoMeasure Image Analysis System (Osteometrics, Decatur, GA) 34,35 following the recommendations of the ASBMR Histomorphometry Nomenclature Committee. 53T12 to L2 vertebrae were processed as LR white plastic-embedded blocks, as we reported previously. 34,353 µm-thick plastic sections were cut using a carbide steel knife on a Shandon Microtome.Sections at the center of the vertebrae were collected to evaluate the dynamic parameters of bone formation using an OsteoMeasure Image Analysis System, as we reported previously, 34,35 following the recommendations of the ASBMR Histomorphometry Nomenclature Committee. 53teoclastogenesis Our culture procedure was modified from our previous reports. 34,35Briefly, we cut open both ends of each femur or tibia to expose the marrow cavity, flushed out BM with 10 mL of α-MEM containing 2% FBS by using a 21-gauge needle, and passed the cells through a 21-G needle 3 times to make single cell suspensions.The cells were incubated in NH 4 Cl solution for 15 min at room temperature to lyse red blood cells.5 × 10 4 cells were seeded in a well of 96-well plates with 5 ng/mL M-CSF for 2 days to generate OCPs.Then RANKL (10 ng/mL) and different inhibitors were added to the cultures for an additional 2-3 days when mature OCs typically are observed under inverted microscopy.The cells were then fixed with 10% neutral, phosphatebuffered formalin for 10 min and stained for TRAP activity.TRAP + cells with 3 or more nuclei were considered as mature OCs.
In vitro osteoblast differentiation assay 1 × 10 6 BM cells from WT mice were seeded in a well of 12-wellplates with α-MEM containing 15% FBS for 5 days to expand the SCs followed by induction of OB differentiation with 25 μg/mL ascorbic acid and 5 mmol/L β-glycerophosphate. 34,35The cells were fixed after 7 d with 10% neutral phosphate-buffered formalin followed by ALP or after 14 d followed by Von Kossa staining to measure the area (Ar.) of ALP + cells and mineralized nodules, respectively.Similarly, bone-derived mesenchymal progenitor cells (BdMPCs) from WT mice 34,35 were used to test the effect of compounds on OB differentiation.Briefly, BdMPCs were seeded in 12-well plates, 1×10 4 /well.From the second day, the cells were induced for OB differentiation, as above, in the presence of different compounds for 7 d.ALP + OB differentiation was evaluated using ALP staining.

Western blot analysis
Macrophages induced by M-CSF from C57BL/6 mouse BM and C3H10T1/2 mouse MSCs, treated with different compounds, were lysed with M-Per mammalian protein extraction reagent (Thermo Dual antiresorptive and anabolic agent Z Yao et al.

Fig. 1
Fig. 1 HABP-HCQ stimulates osteoblast and inhibits osteoclast differentiation.a Synthesis of HABP -Hydrolysis of tetraethyl HABP with bromotrimethylsilane. b C57BL/6 mouse BM cells were cultured with M-CSF for 2 d followed by treatment with RANKL plus the indicated doses of HCQ, HABP-HCQ, or HABP for 3 d.OC number and surface were evaluated after TRAP staining.c BM cells were cultured for 5 d in 24well plates to expand stromal cells, which were induced for OB differentiation in the presence of the indicated doses of HCQ or HABP-HCQ intermittently, 6 h each day, for 5 d.ALP staining was performed to evaluate OB differentiation.d Effects of 1 μmol/L HABP vs. 1 μmol/L HABP-HCQ and HCQ on OB differentiation, tested in 12-well plates, as above in (b).*P < 0.05 & **P < 0.01 vs. vehicle, or #P < 0.01; one-way ANOVA +Dunnett's test

Fig. 2 Fig. 3 2 TbFig. 4 1 (Fig. 5
Fig. 2 HABP-HCQ stabilizes TRAF3 in osteoclast and osteoblast progenitor cells.a C57BL/6 mouse BM cells were cultured with M-CSF for 3 d to expand macrophages, which were treated with vehicle or RANKL plus 3 μmol/L of the indicated compounds for 24 h.Protein levels of TRAF3 and its related level normalized to β-actin were analyzed by WB. b BdMPCs were treated with TGFβ1 plus 3 μmol/L of the indicated compounds for 24 h, and levels of TRAF3 and β-actin protein were analyzed by WB. *P < 0.05 & **P < 0.01 vs. vehicle, #P < 0.01 between the two groups, one-way ANOVA + /Dunnett's test